Visualization of Phosphatidylinositol 3,5-Bisphosphate Dynamics by a Tandem ML1N-Based Fluorescent Protein Probe in Arabidopsis

Phosphatidylinositol 3,5-bisphosphate [PI(3,5)P₂] is a low-abundance phospholipid known to be associated with a wide variety of physiological functions in plants. However, the localization and dynamics of PI(3,5)P₂ in plant cells remain largely unknown, partially due to the lack of an effective fluorescent probe. Using Arabidopsis transgenic plant expressing the PI(3,5)P₂-labeling fluorescent probe (tagRFP–ML1N*2) developed based on a tandem repeat of the cytosolic phosphoinositide-interacting domain (ML1N) of the mammalian lysosomal transient receptor potential cation channel, Mucolipin 1 (TRPML1), here we show that PI(3,5)P₂ is predominantly localized on the limited membranes of the FAB1- and SNX1-positive late endosomes, but rarely localized on the membranes of plant vacuoles or trans-Golgi network/early endosomes of cortical cells of the root differentiation zone. The late endosomal localization of tagRFP–ML1N*2 is reduced or abolished by pharmacological inhibition or genetic knockdown of expression of genes encoding PI(3,5)P₂-synthesizing enzymes, FAB1A/B, but markedly increased with FAB1A overexpression. Notably, reactive oxygen species (ROS) significantly increase late endosomal levels of PI(3,5)P₂. Thus, tandem ML1N-based PI(3,5)P₂ probes can reliably monitor intracellular dynamics of PI(3,5)P₂ in Arabidopsis cells with less binding activity to other endomembrane organelles.